Ratnayake, I. | bioRxiv Science

bioRxiv2019

CellWell: A micropatterned biphasic nanocomposite platform for culturing chondrocytes

Graphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC=\"FIGDIR/small/790030v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (25K):\norg.highwire.dtl.DTLVardef@e0a6c0org.highwire.dtl.DTLVardef@e3b9f7org.highwire.dtl.DTLVardef@c60cb6org.highwire.dtl.DTLVardef@6cb884_HPS_FORMAT_FIGEXP M_FIG C_FIG AbstractWe present a unique micropatterned nanocomposite cell culture platform to model articular cartilage that is suitable for high-throughput single-cell analyses using standard imaging techniques. This platform, the CellWell, is constructed out of a thin, optically transparent substrate that is lithographically micropatterned with a network of wells sized to fit individual cells. The substrate material consists of a thin layer of agarose hydrogel embedded with polyvinyl alcohol nanofibers. The geometries of the wells are designed to reinforce a physiological morphology, thereby combining the physiological advantages of 3D culture systems with the practical advantages of 2D systems. CellWells were found to have compressive moduli of 144 {+/-} 11.5 kPa and 158 {+/-} 0.6 kPa at strain rates of 5 m/s and 15 m/s. The compressive moduli were determined at two different strain rates to allow for comparison of CellWell stiffness with published values of pericellular matrix and with observed values of articular cartilage, which could not be indented at the same rate. Articular chondrocytes seeded in a CellWell were found to maintain their spheroidal morphology more effectively than those seeded in monolayer cultures and to be more easily imaged than those seeded in a 3D scaffold of identical thickness. Through its ease of use and ability to maintain the physiological morphology of chondrocytes, we expect that the CellWell will enhance the clinical translatability of future studies conducted using this culture platform.

bioengineering↗

bioRxiv2019

Notch signaling regulates Akap12 expression and primary cilia length during renal tubule morphogenesis.

Alagille syndrome patients present with loss of function mutations in either JAG1 or NOTCH2. About 40-50% of patients have kidney abnormalities, with multi-cystic, dysplastic kidneys being one of the more frequent kidney defects. Additionally, gain-of-function mutations in NOTCH2 are associated with cystic kidneys in Hajdu-Cheney syndrome patients. Conditional inactivation of Notch1, Notch2, or RBPJ within the nephrogenic lineage impairs nephrogenesis and produces proximal tubule cysts in mice. How perturbations in Notch signaling cause renal tubular cysts remains unclear. Here we have determined that inhibition of Notch signaling in the kidney increases Akap12 expression. Ectopic expression of Akap12 in renal epithelia results in abnormally long primary cilia similar to those observed in Notch-signal-deficiency. Both loss of Notch signaling and elevated Akap12 expression disrupt the ability of renal epithelial cells to form spherical structures with a single lumen when grown embedded in matrix. We conclude that Notch signaling regulates Akap12 expression to ensure normal primary cilia length and renal epithelial morphogenesis, and suggest that diseases associated with defective Notch signaling, such as Alagille syndrome, maybe mechanistically related to ciliopathies.\n\nTranslational StatementThe current study examines how a reduction in Notch signaling results in abnormal renal tubule formation, as occurs in Alagille Syndrome patients with mutations in JAG1 or NOTCH2. The finding that reduced Notch signaling results in abnormally long cilia is suggestive that some of the clinical manifestations in Alagille Syndrome, such as small cystic kidneys, may originate due to defective cilia function. Linking Notch to primary cilia also opens up the possibility that coinheritance of mutations in ciliopathy genes along with a mutation in JAG1 or NOTCH2 may enhance the severity of the clinical phenotypes such as cystic kidney disease and may explain the variable occurrence and onset of kidney disease among Alagille Syndrome patients.

cell biology↗